Protein misfolding and the accumulation of ubiquitylated protein aggregates are hallmarks of a wide-array of human neurodegenerative disorders. The ubiquitin-proteasome system governs proteome surveillance and the selective removal of misfolded proteins. A large source of misfolded proteins arises from defective translation products that are continuously produced during error-prone protein synthesis. Terminal translational stalls resulting from defects within either the mRNA or nascent polypeptide require resolution by co-translational quality control pathways. Despite the importance of maintaining proteome fidelity, translational errors rarely impact the genome and are difficult to detect or measure. Because of these facts, interrogation of mammalian co-translational quality control systems using traditional genetic and molecular approaches is difficult or impossible. As such, there is an enormous gap in our understanding of the ribosome as a quality control platform with regard to how the initial quality control event is sensed and how protein degradation dysfunction is communicated to the protein biogenesis machinery. The contribution of the proposed research is expected bridge this gap in knowledge by characterizing newly identified site-specific regulatory ubiquitylation events on 40S ribosomal proteins that are stimulated by protein homeostasis stress. We further find that this regulatory ubiquitylation of ribosomes is conserved from yeast to man. Additionally, 40S ubiquitylation potentially specifically demarcates a specialized quality control ribosomal subpopulation. We will use a combination of directed molecular and systems-level proteomic approaches to mechanistically define the function of quality control ribosomes as well as identify the molecular complexes that perceive translational stress and catalyze regulatory ribosomal ubiquitylation. Our central hypothesis is that 40S ribosomal ubiquitylation is a required and proximal event in a co-translational quality control pathway that regulates the abundance of defective translation products. The ubiquitylation of ribosomal proteins represents a new molecular feature that can be targeted to manipulate protein homeostasis to potentially modulate and alleviate aging-associated disorders. The objectives within this proposal aim to establish the molecular makeup and mechanism of quality control ribosomes. We will identify specific mRNAs that are enriched or depleted from quality control ribosomes. We will also establish cell lines and animal models that are deficient in 40S regulatory ubiquitylation at specific lysine positions to interrogate the cellular and physiological phenotypes associated with loss of quality control ribosome function. These objectives will set the stage for long-term mechanistic studies detailing cellular strategies aimed at mitigation of translation error and modulation of proteome buffering capacity. The overall goal is to develop targeted strategies to precisely control the balance of protein production and degradation. This would allow for molecular tuning of protein homeostasis to respond to challenges arising, for example, from the accumulation of protein aggregates during the progression of human neurodegenerative diseases.